Regulation of Competence for Genetic Transformation in Streptococcus pneumoniae

University Of Illinois At Chicago, Chicago IL

Investigators

Abstract

Genetic transformation is a naturally occurring, inducible gene replacement process that is relatively simple and very efficient; yet the mechanism of this natural bacterial system for 'gene therapy' is not understood. Competence for genetic transformation in pneumococcus (Streptococcus pneumoniae) occurs during a brief period of highly specialized protein synthesis, coordinated among all cells of an actively growing culture. During a period of approximately 10-20 minutes, the competent cells can transport DNA strands into the cytoplasm and direct them so as to replace homologous genes in the chromosome very efficiently. Central elements of the coordination mechanism are a secreted 17-amino-acid peptide pheromone, a two-component regulatory system that senses the peptide pheromone signal, and a putative accessory sigma factor. To identify genes involved in the pheromone sensing and DNA-processing mechanisms and to determine their roles, this project relies on standard genetic techniques and the efficient gene replacement provided by the natural transformation process itself, in combination with molecular methods in vitro, recombinant DNA cloning using new vectors in pneumococcus, and DNA microarray gene expression analysis. To establish the genetics of the system, gene disruption mutations are made by combining DNA cassettes obtained by polymerase chain reaction synthesis followed by direct insertion into the pneumococcal chromosome. The project will identify and determine the function of genes of this natural transformation system in two broad areas: (a) genes allowing cell-to-cell communication for regulation of this pathway in response to a measure of population density, and (b) genes of the pathway that transports extracellular DNA into the cell and processes it for genetic recombination.